Method for analyzing glycan structure

ABSTRACT

In order to provide an analysis method that is capable of determining a glycan structure with high detection sensitivity, a method of the present invention includes the steps of: carrying out triple quadrupole mass spectrometry at various values of CID energy; creating an energy-resolved profile including yield curves representing relationships between (i) a value of the CID energy and (ii) measured amounts of specific types of product ions; preparing a reference profile, and identifying a glycan structure of a test material by comparing the energy-resolved profile with the reference profile.

This Nonprovisional application claims priority under 35 U.S.C. §119 onPatent Application No. 2012-197908 filed in Japan on Sep. 7, 2012, theentire contents of which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a method for analyzing a glycanstructure. More specifically, the present invention relates to a methodfor analyzing a glycan structure by triple quadrupole mass spectrometry.

BACKGROUND ART

Foreseeing the patent expiry of therapeutic antibodies that are sellingmore than $30 billion worldwide, there is growing interest over howbiosimilar substitutes could win FDA-approval in the near future. Thefirst draft guidance on the evaluation of biosimilarity was published byFDA in February 2012, in which emphasis was placed on the importance ofevaluating minor structural differences that can significantly affectthe potency and safety of biopharmaceuticals, with specific reference toglycosylation patterns, and that such structural characterization beconducted on multiple lots to understand the lot-to-lot variability.

With regard to therapeutic antibodies, which have a commonN-glycosylation consensus sequence at Asn₂₉₇ in the conserved (Fc)region of heavy chain, some specific features of N-glycosylation havebeen characterized to affect potency and safety. For example, absence ofcore fucosylation was demonstrated to enhance antibody-dependentcellular cytotoxicity by 10-fold. Moreover, recent studies revealed thatnon-human oligosaccharide motifs such as glycolylneuraminic acid(Neu5Gc) and galactose-α1,3-galactose (α-Gal epitope) are immunogenicand can cause anaphylaxis in patients expressing specific IgE. Furthercharacterization elucidated more specifically that immunogenicity ofα-Gal epitope was primarily attributed to an extra N-glycan occurringwithin the antigen-binding (Fab) region. These findings have raised theissue of antibody glycosylation to the level that global picture of itsheterogeneity and biological impact is urgently needed.

Here, glycans of glycoproteins are explained. The glycans ofglycoproteins are largely classified into two types of glycans, i.e.,(i) N-glycoside-linked glycans (N-glycans) linked to an asparagineresidue and (ii) O-glycoside-linked glycans (O-glycans) linked toserine, threonine, or the like. The N-glycans have a common corestructure (see the following structural formula) whose terminal linkedto asparagine is referred to as a reducing terminal and whose terminalopposite to the reducing terminal is referred to as a nonreducingterminal.

The N-glycans are classified into (i) high-mannose type having more thanone mannose linked to the nonreducing terminal of the core structure,(ii) complex type having, at the nonreducing terminal, one or moreN-acetylglucosamine (hereinafter referred to as GlcNAc) branches to eachof which galactose, sialic acid, fucose, and the like are linked, and(iii) hybrid type having both a high-mannose type branch and a complextype branch. It is well known that, for example, the complex type andthe hybrid type can have GlcNAc linked to mannose at a branching pointof the core structure (bisecting GlcNAc) and can have fucose linked toGlcNAc at the reducing terminal (core fucose).

Such a structural diversity is observed in a single glycoprotein, and iscalled, for example, Glycoform Heterogeneity. For example, one paperreports that a glycan structure analysis of human serum immunoglobulin Ghaving a single N-glycan binding site revealed that the human serumimmunoglobulin G had 34 types of glycan structures (Non PatentLiterature 1).

As described above, recent studies revealed that differences in glycanstructure significantly affect functions of glycoproteins (see, forexample, Non Patent Literature 2). Accordingly, there are demands for amethod for a quantitative analysis of glycan structures having diversityand highly efficient profiling as to types and proportions of the glycanstructures.

One example of the method for analyzing a glycan structure is a methodof (i) chemically or enzymatically isolating an N-glycan from aglycoprotein, (ii) chemically modifying (labeling) and purifying theN-glycan, and then (iii) detecting the N-glycan by a combination of HPLCand mass spectrometry such as MALDI-TOF MS. This method has advantagessuch as (i) being capable of easily separating labeled glycans accordingto structure by reversed-phase or normal-phase HPLC and (ii) beingcapable of removing impurities through the purification and therebyallowing highly sensitive measurement. On the other hand, this methodhas disadvantages such as (i) requiring complicated pretreatment and(ii) being incapable of obtaining information of each glycosylation sitein a case where the glycoprotein has more than one glycosylation sites.

Another example is a method of (i) breaking a glycoprotein intoglycopeptides, which are peptides to which a glycan is linked, by anenzyme such as trypsin and then (ii) measuring the glycopeptides thusobtained (Patent Literature 1). This measurement is carried out mostlyby use of a mass spectrometer using nano HPLC-ESI as an ion source. Thismass spectrometer makes it possible to not only accumulateglycopeptide-derived signals and quantify glycopeptide but alsodetermine a glycosylation site and estimate a glycan structure throughMS^(n) measurement.

Another paper reports a method of measuring a fragment ion specific toeach glycopeptide with good quantitativity with the use of a triplequadrupole mass spectrometer by a multiple reaction monitoring (MRM)method (Non Patent Literature 3).

CITATION LIST

-   Patent Literature 1-   Japanese Patent Application Publication, Tokukai, No. 2008-309501 A    (Publication Date: Dec. 25, 2008)-   Patent Literature 2-   Japanese Patent Application Publication, Tokukai, No. 2012-58002 A    (Publication Date: Mar. 22, 2012)-   Patent Literature 3-   Japanese Patent Application Publication, Tokukai, No. 2005-265697 A    (Publication Date: Sep. 29, 2005)-   Patent Literature 4-   Japanese Patent Application Publication, Tokukai, No. 2006-145519 A    (Publication Date: Jun. 8, 2006)-   Patent Literature 5-   WO2006/043405 (Publication Date: Apr. 27, 2006)-   Non Patent Literature 1-   Flynn, G. C. et al., Naturally occurring glycan forms of human    immunoglobulins G1 and G2., Mol Immunol, 2010, 47, 2074-2082.-   Non Patent Literature 2-   Shinkawa, T. et al., The absence of fucose but not the presence of    galactose or bisecting N-acetylglucosamine of human IgG1    complex-type oligosaccharides shows the critical role of enhancing    antibody-dependent cellular cytotoxicity., J Biol Chem, 2003, 278,    3466-3473.-   Non Patent Literature 3-   Kurogochi, M. et al., Sialic acid-focused quantitative mouseserum    glycoproteomics by multiple reaction monitoring assay., Mol Cell    Proteomics, 2010, 9, 2354-2368.

SUMMARY OF INVENTION Technical Problem

The conventional methods, however, have problems such as (i) lowsensitivity of detection of glycopeptides in a mixture of peptides withno glycan and the glycopeptides and (ii) difficulty of detecting aglycan structure even by MS/MS analysis of glycopeptides. In addition,since the detection sensitivity of glycopeptides is low, it is not easyto determine a glycan structure.

The present invention was accomplished in view of the above problems,and an object of the present invention is to provide a method foranalyzing a glycan structure with high detection sensitivity.

Solution to Problem

A method of the present invention for analyzing a glycan structure of atest material having a glycan, includes the steps of: (a) measuringspecific types of product ions produced from the test material atvarious values of CID energy by MS/MS; (b) creating an energy-resolvedprofile including yield curves representing relationships between (i)the values of the CID energy and (ii) measured amounts of the respectivespecific types of product ions; (c) preparing a reference profileincluding yield curves representing relationships between (i) the valuesof the CID energy and (ii) measured amounts of respective same types ofproduct ions produced from a reference test material as the specifictypes of product ions, the reference test material being a test materialhaving a glycan and whose structure is known; and (d) identifying theglycan structure of the test material by comparing the energy-resolvedprofile obtained in the step (b) with the reference profile, thespecific types of product ions including at least two types of productions derived from a protonated monosaccharide or disaccharide, and inthe step (a), the measurement by MS/MS being carried out by use of amass spectrometer which causes no Low-mass cutoff.

Advantageous Effects of Invention

According to the present invention, it is possible to determine a glycanstructure of a test material with high detection sensitivity.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a view schematically showing a scheme by which anenergy-resolved yield curve of oxonium ions can be acquired by using atriple quadrupole mass spectrometer.

FIG. 2 is a view showing specific examples of the energy-resolvedoxonium ion profile.

FIG. 3 is a view showing other specific examples of the energy-resolvedoxonium ion profile.

FIG. 4 is a scatter diagram showing a correlation between an optimum CIDenergy value and an m/z value of precursor ions.

FIG. 5 is a view showing a correlation between injection concentrationof a test material to be measured and concentration of an internalstandard.

FIG. 6 is a view showing an MRM chromatogram monitoring oxonium ions ofm/z=138 in digested cetuximab.

FIG. 7 is a view showing an MRM chromatogram obtained from a glycancontaining galactose-1,3-galactose or N-glycolylneuraminic acid motif indigested cetuximab.

FIG. 8 is a view showing energy-resolved oxonium ion profiles of twotypes of glycopeptide containing Lewis×motif.

FIG. 9 is a view showing a result of analysis on lot-to-lotheterogeneity of glycoform of a therapeutic antibody.

FIG. 10 is a view showing an energy-resolved oxonium ion profile of anO-glycopeptide.

FIG. 11 is a view showing glycan structures used for prediction ofidentity of patterns by linear regression analysis.

DESCRIPTION OF EMBODIMENTS

An embodiment of a method of the present invention for analyzing aglycan structure is described below.

A method of the present embodiment for analyzing a glycan structure of atest material having a glycan includes: the step (measuring step) ofmeasuring specific types of product ions produced from the test materialat various values of CID energy by MS/MS; the step (creating step) ofcreating an energy-resolved profile including yield curves representingrelationships between (i) the values of the CID energy and (ii) measuredamounts of the respective specific types of product ions; the step(preparing step) of preparing a reference profile including yield curvesrepresenting relationships between (i) the values of the CID energy and(ii) measured amounts of respective same types of product ions producedfrom a reference test material as the specific types of product ions,the reference test material being a test material having a glycan andwhose structure is known; and the step (identifying step) of identifyingthe glycan structure of the test material by comparing theenergy-resolved profile obtained in the creating step with the referenceprofile, the specific types of product ions including at least two typesof product ions derived from a protonated monosaccharide ordisaccharide, and in the measuring step, the measurement by MS/MS beingcarried out by use of a mass spectrometer which causes no Low-masscutoff.

The inventors of the present invention found that a pattern of anenergy-resolved profile obtained in a similar step to the measuring stepand the creating step is determined by a glycan structure and differsfrom one glycan structure to another. Based on this finding, theinventors of the present invention accomplished the present analysismethod.

The following description deals with an example in which a triplequadrupole mass spectrometer, which causes no Low-mass cutoff, is usedas a mass spectrometer for measurement. Note, however, that the massspectrometer to be used is not limited to this.

(1. Measuring Step)

The measuring step is a step of measuring specific types of product ionsat various values of CID energy by triple quadrupole mass spectrometry.

The term “CID energy” used herein refers to energy which is applied whenions (precursor ions) corresponding to a test material to be measuredare subjected to CID (collision induced dissociation) at a secondquadrupole of the triple quadrupole mass spectrometer. Parameters forregulating the CID energy can vary depending on which device is used. Ingeneral, the “CID energy” refers to a voltage for oscillating ions in acell in which the CID is carried out. The term “product ions” usedherein refers to fragment ions generated as a result of the CID on theprecursor ions. The “m/z” used herein refers to a mass (m) to charge (z)ratio.

It is possible to measure the product ions at various values of the CIDenergy by appropriately setting the CID energy in a range suitable for aused device, but the CID energy is preferably changed as follows:

(i) The CID energy is changed from energy at which a relative intensityof the precursor ions is 100 (i.e., only the precursor ions aredetected) to energy at which the relative intensity of the precursorions is 0.

(ii) The CID energy is changed within a range including an energy pointwhich provides the maximum intensity of each type of product ions.

(iii) The CID energy is changed, for each type of product ions, within arange which includes an energy point which provides the maximumintensity of the each type of product ions and which ranges from minimumenergy for detection of the each type of product ions to energy at whichthe intensity of the each type of product ions is 0.

In the present embodiment, the specific types of product ions to bemeasured are, out of product ions of the test material, product ionsderived from a protonated monosaccharide or disaccharide, and are, forexample, product ions having m/z in a range from 100 to 500. Specificexamples of such product ions encompass product ions having m/z of 163,168, 186, 204, 274, 290, 292, 308, 366, 454, and 470, respectively. Theproduct ion having m/z of 163 corresponds to an oxonium ion of hexose(Hex). The product ion having m/z of 168 corresponds to an oxonium ionof N-acetylhexosamine-2H₂O (HexNAc-2H₂O). The product ion having m/z of186 corresponds to an oxonium ion of N-acetylhexosamine-H₂O(HexNAc-H₂O). The product ion having m/z of 204 corresponds to anoxonium ion of N-acetylhexosamine (HexNAc). The product ion having m/zof 274 corresponds to an oxonium ion of 5-acetylneuraminic acid-H₂O(Neu5Ac-H₂O). The product ion having m/z of 290 corresponds to anoxonium ion of 5-glycolylneuraminic acid-H₂O (Neu5Gc-H₂O). The production having m/z of 292 corresponds to an oxonium ion of5-acetylneuraminic acid (Neu5Ac). The product ion having m/z of 308corresponds to an oxonium ion of 5-glycolylneuraminic acid (Neu5Gc). Theproduct ion having m/z of 366 corresponds to an oxonium ion ofN-acetylhexosamine+hexose (HexNAc+Hex). The product ion having m/z of454 corresponds to an oxonium ion of 5-acetylneuraminic acid+hexose(Neu5Ac+Hex). The product ion having m/z of 470 corresponds to anoxonium ion of 5-glycolylneuraminic acid+hexose (Neu5Gc+Hex).

Note, however, that the product ions to be measured are not limited tothose mentioned above. For example, the product ions to be measured mayinclude a product ion having m/z of 138 and/or a product ion having m/zof greater than 500.

In a case of using an expression “the specific types of product ionsinclude product ions having m/z of 163, 204, 274, and 366,respectively”, the expression means that (i) at least these four typesof product ions having m/z of 163, 204, 274, and 366, respectively, aremeasured and (ii) another/other type(s) of product ions havinganother/other value(s) of m/z may be additionally measured. For example,a product ion having m/z of 138 may be measured in addition to thesefour types of product ions. That is to say, the measuring step may be(i) a step of measuring only the product ions having m/z of 163, 204,274, and 366, respectively, (ii) a step of measuring only the productions having m/z of 138, 163, 204, 274, and 366, respectively, or (iii) astep of measuring another/other type(s) of product ions in addition tothese types of product ions. Note that the product ion having m/z of 138corresponds to an oxonium ion of a fragment of HexNAc.

In the present analysis method, an energy-resolved profile including aplurality of yield curves is created. Accordingly, the product ions tobe measured include at least two types of product ions, preferablyinclude at least two types of product ions selected from the groupconsisting of product ions having m/z of 163, 168, 186, 204, 274, 290,292, 308, 366, 454, and 470, respectively, more preferably include (i)at least one type of product ion selected from the group consisting ofproduct ions having m/z of 163, 168, 186, 274, 290, 292, 308, 366, 454,and 470, respectively and (ii) the product ion having m/z of 204,further more preferably include product ions having m/z of 163, 204,274, and 366, respectively.

As described above, the product ions are measured at various values ofthe CID energy. Note, however, that an amount by which the value of theCID energy is changed is not limited in particular. For example, thevalue of the CID energy can be changed at increments of 3V to 5V.

Use of the triple quadrupole mass spectrometer makes it possible toavoid the following problems arising from Low-mass cutoff. For example,in a case where a mass spectrometer, such as a quadrupole ion trap massspectrometer, which causes Low-mass cutoff is used, sensitivity ofdetection of fragment ions in a low mass range (e.g., m/z=100 to 300) inMS/MS measurement depends on m/z of precursor ions. This means that in acase where, for example, the test material to be measured is aglycopeptide which contains peptides in addition to glycans, m/z of theprecursor ions becomes large, which leads to a problem such as a failureto detect the fragment ions in the low mass range or low sensitivity ofdetection of the fragment ions in the low mass range. Furthermore, insuch a case where the test material to be measured is a glycopeptide,the following problem occurs. Even glycopeptides having an identicalglycan structure can vary in total mass from one another if they aredifferent from one another in amino acid sequence of a peptide. Adifference in m/z of precursor ions results in, due to Low-mass cutoff,a difference in sensitivity of detection of fragment ions in the lowmass range. It is therefore expected that glycopeptides which have anidentical glycan structure but are different from each other in aminoacid sequence are different from each other in obtained yield curve andenergy-resolved profile (later described). It is therefore preferable tocarry out MS/MS measurement with the use of a triple quadrupole massspectrometer, which causes no Low-mass cutoff. Note, however, that amass spectrometer to be used is not limited to a triple quadrupole massspectrometer, provided that it causes no Low-mass cutoff and is capableof successively acquiring MS/MS measurement data on an identicalprecursor ion at various values of the CID energy. The mass spectrometerwhich causes no Low-mass cutoff can be any mass spectrometer whosesensitivity of detection of fragment ions in a low mass range (e.g.,m/z=100 to 300) does not depend on m/z of precursor ions.

Examples of a method for ionization encompass FAB (fast atombombardment), CI (chemical ionization), ESI (electrospray ionization),MALDI (matrix-assisted laser desorption/ionization), and APCI(atmospheric pressure chemical ionization). Above all, ESI is suitablyused. Examples of ESI encompass microspray ionization and nanosprayionization. Of the two examples of ESI, the nanospray ionization issuitably used.

The test material to be analyzed in the present analysis method is notlimited in particular, provided that it is a test material which has aglycan and can be subjected to triple quadrupole mass spectrometry.Suitably, the test material to be analyzed is a glycoprotein or aglycopeptide. The glycoprotein and the glycopeptide each may be oneobtained from a biological tissue, a biological cell, or the like or maybe one obtained by adding a glycan to an artificially synthesizedprotein or peptide. The glycopeptide may be one obtained through acidhydrolysis or enzymatic decomposition of a glycoprotein. In a case wherethe glycopeptide is obtained through decomposition of a glycoprotein,the number of glycosylation sites of the glycoprotein is not limited to1, but the glycoprotein may have more than 1 glycosylation site.Further, in the case where the glycopeptide is obtained throughdecomposition of a glycoprotein, it is preferable to use a glycopeptidethat has been subjected to separation and purification by HPLC or thelike. The glycoprotein and the glycopeptide each may have an N-glycan ormay have an O-glycan. N-glycans, which have the common core structure,are expected to have the smaller number of variations of theenergy-resolved profile (later described) than O-glycans. It istherefore easier to identify a glycan structure of an N-glycan than aglycan structure of an O-glycan. The present method is therefore moresuitably applied to a glycoprotein having an N-glycan or a glycopeptidehaving an N-glycan.

(2. Creating Step)

The creating step is a step of creating an energy-resolved profile(referred to also as an energy-resolved oxonium ion profile) whichincludes yield curves representing relationships between (i) the CIDenergy and (ii) measured amounts of the respective specific types ofproduct ions.

Each of the yield curves (referred to also as energy-resolved yieldcurves) representing relationships between the CID energy and measuredamounts of respective types of product ions can be created, for example,by plotting measured amounts (counted values) (represented by a y-axis)of a target product ion against values of the CID energy (represented byan x-axis). The measured amounts of the target product ion which are tobe plotted may be relative amounts to another type of product ion or maybe a ratio to the total counted values of all the measured product ions.In one aspect of the present embodiment, yield curves are created insuch a manner that (i) a product ion having m/z of 138 is measured and(ii) a measured amount of the target product ion is normalized with theuse of a measured amount of this product ion. More specifically, in themeasuring step, the product ion having m/z of 138 is measured inaddition to the other specific types of product ions. A value of the CIDenergy at which a measured amount of this product ion becomes maximum isdetermined. Then, the measured amount of this product ion at the valuethus determined is assumed to be a standard value 100. Measured amountsof the other specific types of product ions are expressed as relativevalues to the standard value. The measured amounts of the other specifictypes of product ions are thus normalized.

The energy-resolved profile is a profile, for a single test material,which combines (unifies) yield curves of the respective specific typesof product ions. That is, in a case where four specific types of productions are measured, the energy-resolved profile includes four yieldcurves.

(3. Preparing Step)

The preparing step is a step of preparing a reference profile whichincludes yield curves representing relationships between (i) a value ofthe CID energy and (ii) measured amounts of respective same types ofproduct ions produced from a reference test material as the specifictypes of product ions measured in the measuring step, which referencetest material is a test material which has a glycan and whose structureis known.

The expression “preparing a reference profile” used herein encompassesnot only a case where a reference profile is newly created, but also acase where a reference profile that has been already created is madeavailable. The expression “made available” means, for example, obtaininga reference profile that has been already created or allowing an accessto a server or the like in which the reference profile is stored. The“reference profile that has been already created” may be one created byanother person. The “same types of product ions” refer to types ofproduct ions having same values of m/z as the specific types of productions measured in the measuring step.

The reference profile is same in nature as the energy-resolved profile,and is obtained from a reference test material whose structure includinga glycan portion is known. The term “structure” used herein refers to,in the case of a glycan, a sequence of the glycan and positions at whichsugars are linked, and refers to, in the case of a protein or a peptide,an amino acid sequence thereof and positions at which glycans arelinked.

The reference test material is a test material of the same type as thetest material to be analyzed in the present analysis method. That is, ina case where the test material to be analyzed is a glycopeptide, thereference test material is a glycopeptide as well. Further, in a casewhere the test material to be analyzed is a glycopeptide having anN-glycan, the reference test material is a glycopeptide having anN-glycan as well. Note, however, that even in such cases where the testmaterial to be analyzed and the reference test material areglycopeptides, the test material to be analyzed and the reference testmaterial need not be same in amino acid sequence of a peptide.

The reference profile can be created according to the methods used inthe measuring step and the creating step. Specifically, triplequadrupole mass spectrometry is carried out while changing a value ofthe CID energy so that, out of types of product ions produced from thereference test material, the same types of product ions as the types ofthe specific types of product ions are measured at various values of theCID energy. Then, yield curves are created which represent relationshipsbetween (i) the value of the CID energy and (ii) measured amounts of therespective types of the product ions thus measured. Then, a referenceprofile combining these yield curves is created. It is only necessarythat the types of product ions measured for creation of the referenceprofile include the same types of product ions as the specific types ofproduct ions. That is, another/other type(s) of product ions havinganother/other value(s) of m/z may be additionally measured. In a casewhere the yield curves included in the energy-resolved profile of thetest material to be analyzed are normalized, the yield curves includedin the reference profile are normalized as well.

The reference profile thus obtained is associated with the test materialwhich was used for creation of the reference profile and whose structureis known. That is, a reference profile is associated with a glycanstructure of a test material used for creation of the reference profile.One reference profile can be distinguished from another according to adifference in pattern of yield curves included therein. That is,differences in glycan structure among test materials result in creationof reference profiles of different patterns.

In the identifying step (described later), the energy-resolved profileof the test material to be analyzed is compared with reference profilesthus prepared, and in a case where a reference profile that is identicalto the energy-resolved profile is found, the glycan structure of thetest material to be analyzed is identified as a glycan structureassociated with the reference profile thus found. That is, whether aglycan structure can be identified or not in the identifying stepdepends on whether a reference profile associated with the glycanstructure is prepared or not. It is therefore preferable to preparereference profiles for a larger number of glycan structures.

(4. Identifying Step)

The identifying step is a step of identifying a glycan structure of thetest material to be analyzed by comparing the energy-resolved profileobtained in the creating step with the reference profile.

Specifically, the energy-resolved profile of the test material to beanalyzed which energy-resolved profile is obtained in the creating stepis compared with prepared reference profiles. In a case where areference profile that is identical to the energy-resolved profile isfound, the glycan structure of the test material to be analyzed isidentified as a glycan structure associated with the reference profilethus found. In a case where no reference profile that is identical tothe energy-resolved profile is found, the glycan structure of the testmaterial to be analyzed is identified as any of glycan structures otherthan the glycan structures associated with the prepared referenceprofiles.

A method for determining whether the energy-resolved profile isidentical to a reference profile or not is not limited to a specificone, and can be a known pattern recognition technique. Examples of theknown pattern recognition technique encompass a prediction approachusing linear regression analysis.

(5. Quantifying Step)

In addition to the above steps, the method of the present embodiment foranalyzing a glycan structure further includes the step (quantifyingstep) of quantifying the test material with the use of a standardmaterial whose concentration is known.

The quantifying step is a step of quantifying the test material to beanalyzed by comparing (i) a measured amount of a product ion having m/zof 138 produced from the test material to be analyzed and (ii) ameasured amount of a product ion having m/z of 138 produced from thestandard material whose concentration is known. It is preferable thatthe product ion having m/z of 138 be measured at CID energy at whichgeneration efficiency of this product ion becomes maximum. Theexpression “generation efficiency becomes maximum” used herein alsoencompasses a case where generation efficiency is estimated to becomemaximum. The concentration of the standard material can be measured inadvance by a known quantification technique such as UV absorbancemeasurement. In a case where the test material to be analyzed is aglycopeptide, the standard material is preferably a glycopeptide aswell, more preferably a glycopeptide having an identical amino acidsequence to that of the glycopeptide of the test material to beanalyzed.

As demonstrated in Examples that will be described later, there is anextremely strong linear correlation between (i) a value of the CIDenergy at which generation efficiency of the product ion of m/z=138becomes maximum and (ii) m/z of glycopeptides. It is therefore possibleto obtain a value of the CID energy at which generation efficiency ofthe product ion of m/z=138 produced from the test material to beanalyzed becomes maximum by multiplying m/z of the test material to beanalyzed by a coefficient x/y where x is a value of the CID energy atwhich generation efficiency of the product ion of m/z=138 produced fromthe reference test material whose m/z is known becomes maximum and y ism/z of the reference test material. The value of the CID energy at whichgeneration efficiency of the product ion of m/z=138 produced from thereference test material whose m/z is known becomes maximum can be easilyobtained by measuring the product ion of m/z=138 produced from thereference test material at various values of the CID energy. In a casewhere the reference test material has an identical structure to that ofthe test material to be analyzed, it is possible to carry out thequantification more accurately.

Alternatively, the value of the CID energy at which generationefficiency of the product ion of m/z=138 produced from the test materialto be analyzed becomes maximum can be obtained based on (i) m/z of thetest material to be analyzed and (ii) a calibration curve which iscreated by carrying out linear regression analysis with the use ofvalues of the CID energy, for respective plural reference test materialswhose m/z is known, at which generation efficiency of the product ion ofm/z=138 becomes maximum.

As described above and as demonstrated in Examples that will bedescribed later, the method of the present invention for analyzing aglycan structure is an extremely versatile method which is capable ofselectively measuring a target substance, in a test material, having aglycan with high sensitivity. Accordingly, the method of the presentinvention for analyzing a glycan structure is applicable to experimentsand researches in general targeted at a glycan. In industrial fields,there are high demands for a quality control test for glycan structuresof biotechnology-based drugs which have formed a huge market. Therefore,wide ranging applications of the present invention are expected also inthe industrial fields.

(Summary)

As described above, a method of the present invention for analyzing aglycan structure of a test material having a glycan, includes the stepsof: (a) measuring specific types of product ions produced from the testmaterial at various values of CID energy by MS/MS; (b) creating anenergy-resolved profile including yield curves representingrelationships between (i) the values of the CID energy and (ii) measuredamounts of the respective specific types of product ions; (c) preparinga reference profile including yield curves representing relationshipsbetween (i) the values of the CID energy and (ii) measured amounts ofrespective same types of product ions produced from a reference testmaterial as the specific types of product ions, the reference testmaterial being a test material having a glycan and whose structure isknown; and (d) identifying the glycan structure of the test material bycomparing the energy-resolved profile obtained in the step (b) with thereference profile, the specific types of product ions including at leasttwo types of product ions derived from a protonated monosaccharide ordisaccharide, and in the step (a), the measurement by MS/MS beingcarried out by use of a mass spectrometer which causes no Low-masscutoff.

According to the arrangement, an energy-resolved profile for a testmaterial to be measured is created. The energy-resolved profilerepresents relationships between (i) CID energy in MS/MS measurement and(ii) measured amounts of respective specific types of product ions. Theenergy-resolved profile thus obtained is compared with a referenceprofile that is separately prepared. The reference profile is obtainedfrom a test material whose structure is known. Accordingly, in thereference profile, an energy-resolved profile representing relationshipsbetween (i) CID energy in MS/MS measurement and (ii) measured amounts ofrespective specific types of product ions is associated with a glycanstructure. It is therefore possible to identify the test material to beanalyzed by comparing the energy-resolved profile for the test materialto be analyzed and the reference profile. The product ions to bemeasured are product ions derived from a protonated monosaccharide ordisaccharide, i.e., oxonium ions derived from a glycan. Therefore,measurement can be carried out irrespective of a structure other than aglycan part to be analyzed. Note that most of the product ions derivedfrom a protonated monosaccharide or disaccharide are in a low mass rangefrom 100 m/z to 500 m/z. According to the present analysis method, theMS/MS measurement is carried out by use of a mass spectrometer whichcauses no Low-mass cutoff. This makes it possible to measure productions with high detection sensitivity without the need for a process forseparating a glycan from the test material to be analyzed. Since aglycan is not separated in advance, it is possible to, even in a casewhere there are a plurality of precursor ions having respective glycanstructures, distinguish product ions derived from the respectiveprecursor ions.

The method of the present invention for analyzing a glycan structure ispreferably arranged such that the specific types of product ions includeat least two types of product ions selected from the group consisting ofproduct ions having m/z of 163, 168, 186, 204, 274, 290, 292, 308, 366,454, and 470, respectively. The method of the present invention foranalyzing a glycan structure is more preferably arranged such that thespecific types of product ions include (i) at least one type of production selected from the group consisting of product ions having m/z of163, 168, 186, 274, 290, 292, 308, 366, 454, and 470, respectively and(ii) a product ion having m/z of 204.

The method of the present invention for analyzing a glycan structure ispreferably arranged such that the specific types of product ions includeproduct ions having m/z of 163, 204, 274, and 366, respectively.

The method of the present invention for analyzing a glycan structure ispreferably arranged such that the specific types of product ions furtherinclude a product ion having m/z of 138; and in the step (b), theenergy-resolved profile is created by normalizing the yield curves withuse of a measured amount of the product ion having m/z of 138.

The method of the present invention for analyzing a glycan structure ispreferably arranged such that in the step (a), the measuring isperformed by using a sample in which a standard material which has aglycan and whose concentration is known is added in addition to the testmaterial; the specific types of product ions further include a production having m/z of 138; and the method further includes the step of (e)quantifying the test material by comparing (i) a measured amount of theproduct ion having m/z of 138 produced from the test material and (ii) ameasured amount of the product ion having m/z of 138 produced from thestandard material.

The method of the present invention for analyzing a glycan structure ispreferably arranged such that in the step (e), the test material isquantified on basis of (i) the measured amount of the product ion havingm/z of 138 produced from the test material and (ii) the measured amountof the product ion having m/z of 138 produced from the standardmaterial, each of which measured amounts are obtained at a value of theCID energy at which the product ion having m/z of 138 becomes maximum.

The method of the present invention for analyzing a glycan structure ispreferably arranged such that the value of the CID energy at which theproduct ion having m/z of 138 becomes maximum is a value estimated basedon a calibration curve and a value of m/z of a precursor ion of the testmaterial to be analyzed, the calibration curve being created by (I)measuring, at various values of the CID energy, in advance the production having m/z of 138 in a plurality of test materials each having aglycan by MS/MS and then (II) carrying out linear regression analysiswith use of (i) values of the CID energy at which measured amounts ofthe product ion having m/z of 138 in the plurality of test materialsbecome maximum and (ii) values of m/z of precursor ions of the pluralityof test materials.

The method of the present invention for analyzing a glycan structure ispreferably arranged such that in the step (a), the measurement by MS/MSis carried out by use of a triple quadrupole mass spectrometer.

The method of the present invention for analyzing a glycan structure ispreferably arranged such that the test material is a glycopeptide.

The method of the present invention for analyzing a glycan structure ispreferably arranged such that the glycopeptide is a glycopeptide havingan N-glycan.

The method of the present invention for analyzing a glycan structure ispreferably arranged such that the glycopeptide is a glycopeptide havingan O-glycan.

The embodiment of the present invention will be described below in moredetail with reference to Examples below. The present invention is notlimited to Examples below, but details can be changed in variousmanners. The present invention is not limited to the description of theembodiments above, but may be altered by a skilled person within thescope of the claims. An embodiment based on a proper combination oftechnical means disclosed in different embodiments is encompassed in thetechnical scope of the present invention. Further, all of the documentsdescribed in this specification are incorporated herein by reference

EXAMPLES Example 1 Identification of Glycan Structure with Use ofEnergy-Resolved Oxonium Ion Profile

FIG. 1 shows a scheme by which an energy-resolved yield curve of oxoniumions is acquired by using a triple quadrupole mass spectrometer. Thefollowing overviews this scheme. First, a glycoprotein test material(immunoglobulin in FIG. 1) is digested by trypsin into apeptide/glycopeptide mixture. The mixture thus obtained is fractionatedby HPLC, and is then subjected to mass spectrometry using a triplequadrupole mass spectrometer. In the triple quadrupole massspectrometer, a first quadrupole (Q1) isolates glycopeptide ions withunique mass (filtering), which are subsequently guided into a secondquadrupole (Q2) where they undergo CID. The kinetic energy of precursorions, which governs energy and rate of the CID in Q2, can be controlledby changing an electrode potential (corresponding to CID energy) of ionentry into Q2. A third quadrupole (Q3) filters product ions toselectively detect oligosaccharide-derived oxonium ions (see the frame Aof FIG. 1) at high-sensitivity. The electrode potential of ion entryinto Q2 is changed in stepwise fashion in repetitive measurements ofshort time intervals for each mass filter settings in Q3. In this way,energy-resolved yield curves whose horizontal axes represent anelectrode potential of ion entry into Q2 and whose vertical axesrepresent measured ion intensity are obtained for respective types ofoxonium ions (see the frame B of FIG. 1). Use of a MRM (MultipleReaction Monitoring) mode makes it possible to simultaneously acquireenergy-resolved yield curves of respective types of oxonium ions.Energy-resolved yield curves of respective types of oxonium ionsobtained for a particular type of precursor ions are collectivelyreferred to as an “energy-resolved oxonium ion profile”.

Next, the following describes a specific example of how anenergy-resolved oxonium ion profile is created.

The following example attempted to create energy-resolved oxonium ionprofiles of various glycopeptides prepared from IgG molecule, whichshare the same amino acid sequence (EEQYNSTYR: SEQ ID NO: 1) but beardifferent glycan structures.

The glycopeptides were measured by 4000 QTRAP triple quadrupole massspectrometer (AB Sciex, Foster City, Calif.) with Agilent 1200 nano-HPLCsystem (Agilent Technologies, Palo Alto, Calif.). In this example,specific types of oxonium ions (m/z=138, 163, 204, 274, and 366) weresimultaneously measured by the multiple reaction monitoring mode. First,IgG tryptic digest, column-separated at a flow rate of 250 mL/min with achip-integrated capillary column having an inside diameter of 75 μmmanufactured by Nikkyo Technos, Co., Ltd. (Tokyo, Japan), was introducedto an ion source. An ion spray voltage for nanospray was set to 2200V,and a nitrogen gas was used as a curtain gas (12 psi) and a collisiongas. The mass resolution of the first quadrupole (Q1) was set in a HIGHmode, the mass resolution of the third quadrupole (Q3) was set in a LOWmode, and a pause between measurements was set to 2 milliseconds. As forconditions for the CID, device parameters of CAD=4 was used, anddeclustering potential and entrance potential were set to 70V and 10V,respectively. The multiple reaction monitoring assay was carried out fora large number of transitions, i.e., combinations of (i) a setting valueof Q1 (m/z of a target glycopeptide), (ii) a setting value of Q3(138.05, 163, 204, 274, or 366), and (iii) a value of the CID energywhich is changed by increments of 3V or 5V (since a setting value ofmass at one value of the CID energy cannot be the same as that atanother value of the CID energy due to software specification, thesetting value of mass is, strictly speaking, changed by 0.001 as thevalue of the CID energy is changed, but the difference in the settingvalues is insignificant in the measurement). Further, the ion extractiontime (Dwell Time) for each transition was set to be within a range from20 milliseconds to 50 milliseconds so that a measurement cycle time fora single measurement target falls within around 1 second.

Energy-resolved yield curves of the respective types of oxonium ionswere obtained by normalizing measured amounts of the respective types ofoxonium ions with respect to the highest signal intensity (=100) of theenergy-resolved yield curve of the oxonium ion of m/z=138. Althoughother larger-fragment oxonium ions were measured as well, plotting ofsuch oxonium ions was omitted for simplification unless a relativeamount of such oxonium ions exceeds 5%.

(a) through (d) of FIG. 2 show some examples of energy-resolved oxoniumion profiles thus created. Note that (a) through (d) of FIG. 2 eachcollectively show energy-resolved oxonium ion profiles of a respectiveplurality of glycan structures instead of showing an energy-resolvedoxonium ion profile of a single glycan structure. In the schematicglycan structures shown above the energy-resolved oxonium ion profiles,the filled squares each represent N-acetylglucosamine (GlcNAc), thefilled circles each represent mannose (Man), the open circles eachrepresent galactose (Gal), the inverted triangles each represent fucose(Fuc), and the rhombuses each represent 5-acetylneuraminic acid(Neu5Ac). In (a) through (d) of FIG. 2, the energy-resolved yieldcurves, which correspond to the respective types of oxonium ions havingrespective values of m/z, are each indicated by a line whose thicknessis almost same as that of a rectangular frame showing m/z of acorresponding type of oxonium ion.

(a) of FIG. 2 collectively shows energy-resolved oxonium ion profiles ofthree types of glycopeptides each having a non-galactosylated glycanstructure. (b) of FIG. 2 collectively shows energy-resolved oxonium ionprofiles of three types of glycopeptides each having a high-mannose typeglycan structure. (c) of FIG. 2 collectively shows energy-resolvedoxonium ion profiles of two types of glycopeptides each having a hybridtype glycan structure. (d) of FIG. 2 collectively shows energy-resolvedoxonium ion profiles of two types of glycopeptides each having asialylated glycan structure.

As shown in (a) of FIG. 2, measured amounts of the oxonium ion ofm/z=204 (GlcNAc) in the glycopeptides each having a non-galactosylatedglycan structure were apparently greater than those of the oxonium ionof m/z=204 in other glycopeptides having the same number of GlcNAc (see,for example, (c) or (d) of FIG. 2). It is hypothesized that this isbecause the precursor ions are protonated so that GlcNAc becomes aleaving group.

As shown in (b) of FIG. 2, measured amounts of the oxonium ion ofm/z=163 (hexose) in the glycopeptides each having the high-mannose typeglycan structure were apparently greater than those in otherglycopeptides. Since almost no oxonium ion of m/z=163 was measured inthe glycopeptides each having the hybrid type glycan structure ((c) ofFIG. 2), the high yield of the oxonium ion of m/z=163 in theglycopeptides each having the high-mannose type glycan structure cannotbe explained by the number of mannosyl linkages alone. A possibleexplanation for such a difference is that mannose or galactose which isin proximity to GlcNAc lacks a labile proton needed for oxonium ionformation.

As shown in (a) through (d) of FIG. 2, the glycopeptides exhibitedenergy-resolved oxonium ion profiles that can be distinguished from oneanother although a characteristic pattern could be observed for eachtype of glycan structure (non-galactosylated type, high-mannose type,hybrid type, or sialylated type).

This analysis revealed that even glycopeptide isoforms each of which hasa glycan structure of 4[Hex]4[GlcNAc]1[Fuc] and which are different onlyin linkage position of terminal galactose (one of them has terminalgalactose at a α1-3 branch, whereas the other one of them has terminalgalactose at a α1-6 branch can be distinguished from each other (FIG.3). As shown in FIG. 3, these isoforms were different at least inenergy-resolved yield curve of the oxonium ion of m/z=366. That is, theisoform having terminal galactose at the α1-6 branch was higher in yieldof the oxonium ion of m/z=366 at low electrode potentials than theisoform having terminal galactose at the α1-3 branch. In a case where amixture test material of these isomers (mixed at a ratio of 1:1 or 1:2)was measured, a yield curve of the oxonium ion of m/z=366 wasconstructed between the yield curves of the oxonium ion of m/z=366 thatare obtained by analyzing these isomers separately. This shows that useof the present analysis method utilizing an energy-resolved oxonium ionprofile makes it possible to predict an abundance ratio of structuralisomers in a mixture test material without the need to separate theisomers by chromatography. Note that the same explanation as that forthe schematic glycan structures shown in FIG. 2 can be applied to theschematic glycan structures shown in FIG. 3.

Example 2 Prediction Using Linear Regression Analysis

In Example 2, model data was obtained by subjecting test materials whoseglycan structures are known to similar measurement and data processingto those in Example 1 and then linear regression analysis using thismodel data was carried out.

First, two types of glycopeptides having the glycan structures shown in(a) and (b) of FIG. 11, respectively, were prepared, and measured valuesof respective types of product ions at each value of CID energy wereobtained. These glycopeptides are identical in mass but are differentfrom each other in structure. For convenience, the glycan structureshown in (a) of FIG. 11 was set to 0 and the glycan structure shown in(b) of FIG. 11 was set to 1. These values 0 and 1 were used as anobjective variable Y for specifying a type of a glycan structure. Notethat the same explanation as that for the schematic glycan structuresshown in FIG. 2 can be applied to the schematic glycan structures shownin FIG. 11. Normalized intensities (measured values) of the respectivetypes of product ions measured at each value of CID energy were used asa dependent variable X. Specifically, the linear regression analysis wascarried out by measuring each of the glycan structures ten times whileusing, as the dependent variable, a total of 24 measured values, i.e.,(i) measured values of a product ion of m/z=204 at 12 points of CIDenergy (24, 27, 30, 33, 36, 39, 42, 45, 50, 55, 60, and 65 eV) and (ii)measured values of a product ion of m/z=366 at 12 points of CID energy(24, 27, 30, 33, 36, 39, 42, 45, 50, 55, 60, and 65 eV).

As a result, the following estimation equation was obtained:

Y=−1.64X ₁+0.817X ₂+0.345X ₃+0.284X ₄+7.138X ₅+0.297X ₆−0.384

where X₁ through X₆ represent a measured value of the product ion ofm/z=366 at CID energy of 24 eV, a measured value of the product ion ofm/z=204 at CID energy of 27 eV, a measured value of the product ion ofm/z=204 at CID energy of 36 eV, a measured value of the product ion ofm/z=204 at CID energy of 42 eV, a measured value of the product ion ofm/z=366 at CID energy of 65 eV, and a measured value of the product ionof m/z=204 at CID energy of 39 eV, respectively. If the data of theglycan structure shown in (a) of FIG. 11 is assigned to the estimationequation, Y becomes a value close to 0, whereas if data of the glycanstructure shown in (b) of FIG. 11 is assigned to the estimationequation, Y becomes a value close to 1. Obtained values of Y which fallwithin a confidence interval of 95% of this model can be determined assignificant judgment results.

Other plural types (three types) of test materials each having anidentical glycan structure to that shown in (b) of FIG. 11 were eachmeasured twice, and a value of Y was obtained for each of these types oftest materials. As a result, two types of test materials each exhibitedan estimated value of Y which was close to 1 and was within the 95%confidence interval. Therefore, these two types of test materials can bejudged to have the glycan structure shown in (b) of FIG. 11.

Next, data of a test material having a glycan structure different fromboth of the glycan structure shown in (a) of FIG. 11 and the glycanstructure shown in (b) of FIG. 11 was obtained and assigned to theestimation equation. As a result, this test material exhibited anestimated value of approximately 2. This result showed that this testmaterial had a structure different from the glycan structure shown in(a) of FIG. 11 and the glycan structure shown in (b) of FIG. 11.

Example 3 High-Sensitivity Quantification of Glycopeptides

An observation of various energy-resolved oxonium ion profiles revealedthat energy-resolved yield curve shapes of the oxonium ion of m/z=138were similar among tested glycan structures. To clarify this, thefollowing test was conducted.

(Materials)

As human immunoglobulin (IgG) test materials, Cetuximab was purchasedMerck Serono (Tokyo, Japan) and Trastuzumab was purchased from ChugaiSeiyaku (Osaka, Japan). These test materials were digested by trypsin.As a result, standard materials containing a large variety ofglycopeptides were obtained. Specifically, 80 μg of each of these testmaterials was dissolved in 33 μL of 8 M urea solution. To the solutionthus obtained, 1.7 μL of a dithiothreitol-triethylammonium bicarbonatesolution (100 mM dithiothreitol, 1 M triethylammonium bicarbonate) wasadded. The mixture was reduced at 55° C. for 30 minutes. Next, afteraddition of 3.5 μL of 0.5M iodoacetate, the mixture was alkylated atroom temperature in a dark place for 30 minutes, and was then dilutedwith 300 μL of a dithiothreitol-triethylammonium bicarbonate solution(2.5 mM dithiothreitol, 25 mM triethylammonium bicarbonate). Thereto, 4μg of lysyl endopeptidase (Wako Pure Chemical Industries, Ltd.) wasadded, and after 2 hours of reaction at 37° C., 4 μg of Trypsin Gold(Promega KK) was added. After 4 hours of digestion reaction, the mixturewas diluted with 700 μL of a 3% acetonitrile solution, and was thensubjected to desalting purification with the use of an OASIS HLBsolid-phase extraction cartridge (Nihon Waters K.K.). Here, aflow-through fraction and a 15% acetonitrile elusion fraction wereseparately collected. The flow-through fraction and the elusion fractionwere diluted with a 0.1% acetic acid solution twofold and fivefold,respectively. These fractions were used as standard measurementmaterials. The flow-through fraction contained a glycopeptide grouphaving the amino acid sequence EEQYNSTYR (SEQ ID NO: 1), and the 15%acetonitrile elusion fraction contained a glycopeptide group having theamino acid sequence MNSLQSNDTAIYYCAR (SEQ ID NO: 2) (only Cetuximab).

(Device Settings and Measurement Method)

The measurement test materials were measured by the MRM mode of 4000QTRAP triple quadrupole mass spectrometer (AB Sciex, Foster City,Calif.) with nano-HPLC system. Conditions for the measurement weresimilar to those in Example 1. Note, however, that the multiple reactionmonitoring assay was carried out for a large number of transitions,i.e., combinations of (i) a setting value of Q1 (m/z of a targetglycopeptide), (ii) a setting value of Q3 (fixed to 138.05), and (iii) avalue of the CID energy which is changed by increments of 3V or 5V(since a setting value of mass at one value of the CID energy cannot bethe same as that at another value of the CID energy due to softwarespecification, the setting value of mass is, strictly speaking, changedby 0.001 as the value of the CID energy is changed, but the differencein the setting values is insignificant in the measurement).

(Data Processing Method)

Measured data was loaded with analysis software MultiQuant ver.2.02, andwas subjected, for each transition, to peak integration of masschromatogram under a condition of a smoothing width of 1 point. Theintegrated values were exported to spreadsheet software, and a table inwhich CID energy setting values and the integrated values are associatedwith one another was created. In this table, integrated values for eachtransition were normalized as percentage relative to the maximumintegrated value in the data set. Each glycopeptide was measured threetimes, and an average of normalized integrated values was obtained.Based on the data thus obtained, CID energy (optimum CID energy) atwhich generation efficiency of the product ion of m/z=138 became maximumwas obtained for each glycopeptide. The optimum CID energy was definedas a CID energy setting value of a transition in which the average peakintegrated value is closest to 100. Note, however, that in a case wherethe same integrated value falling in a range from 98 to 100 wassuccessively obtained, an average of CID energy values associated withthese integrated values was used as the optimum CID energy. The resultis shown in Table 1.

TABLE 1 Constituents of N-glycan The number The The The Optimum Sampleof number number number CID NO Amino acid sequence HexNAc of Hex of Fucof SA m/z energy 1 EEQYNSTYR 3 3 1 0 811.4 75 2 EEQYNSTYR 3 4 0 0 816.775 3 EEQYNSTYR 3 4 1 0 865.4 80 4 EEQYNSTYR 3 4 0 1 913.8 95 5 EEQYNSTYR3 5 1 0 919.4 90 6 EEQYNSTYR 4 3 0 0 830.3 80 7 EEQYNSTYR 4 3 1 0 878.182.5 8 EEQYNSTYR 4 4 0 0 884.4 85 9 EEQYNSTYR 4 4 0 1 981.4 90 10EEQYNSTYR 4 4 1 0 933.1 90 11 EEQYNSTYR 4 4 1 1 1030.1 105 12 EEQYNSTYR4 5 0 0 938.4 95 13 EEQYNSTYR 4 5 1 0 987.1 95 14 EEQYNSTYR 4 5 0 11035.5 105 15 EEQYNSTYR 4 5 1 1 1084.2 100 16 EEQYNSTYR 5 3 0 0 898.1 9017 EEQYNSTYR 5 3 1 0 952.1 100 18 EEQYNSTYR 5 4 0 0 946.8 90 19EEQYNSTYR 5 4 1 0 1000.8 92.5 20 MNSLQSNDTAIYYCAR 5 8 1 0 1092.5 109 21MNSLQSNDTAIYYCAR 5 9 1 0 1133.1 110 22 MNSLQSNDTAIYYCAR 4 7 1 0 1001.395 23 MNSLQSNDTAIYYCAR 4 6 2 0 997.3 100 24 MNSLQSNDTAIYYCAR 4 6 1 0960.7 91 25 MNSLQSNDTAIYYCAR 4 5 1 1 997.1 100 26 MNSLQSNDTAIYYCAR 4 4 10 879.7 86 27 MNSLQSNDTAIYYCAR 4 5 1 0 920.2 97 28 MNSLQSNDTAIYYCAR 4 31 0 839.2 80 HexNAc: N-acetylhexosamine, Hex: hexose (6-carbon sugar),Fuc: fucose, SA: sialic acid

Based on the result shown in Table 1, a scatter diagram was created byplotting m/z of glycopeptides in an X-axis direction and optimum CIDenergy values in a Y-axis direction (FIG. 4). In FIG. 4, the circleseach represent data of a glycopeptide containing three GlcNAc, thesquares each represent data of a glycopeptide containing four GlcNAc,and the triangles each represent data of a glycopeptide containing fiveGlcNAc. Out of these marks, the open ones each represent data of aglycopeptide whose peptide linked with a glycan has the amino acidsequence MNSLQSNDTAIYYCAR (SEQ ID NO: 2), and the filled ones eachrepresent data of a glycopeptide whose peptide linked with a glycan hasthe amino acid sequence EEQYNSTYR (SEQ ID NO: 1). As shown in FIG. 4,this scatter diagram suggested a linear correlation between m/z of theglycopeptides and the optimum CID energy values. As a result ofcollinear approximation by linear regression, there observed anextremely strong correlation having a multiple correlation coefficient(R²) of 0.842. All the points, which are plotted on the scatter diagramso as to be distinguished from one another according to differences inthe number of GlcNAc and in amino acid sequence, are located in thevicinity of an approximate curve. This shows that factors such as thedifferences in the number of HexNAc and in amino acid sequence do notinfluence the correlation. The fact that a glycan structure, an aminoacid sequence of a peptide, and the charge number do not influence acorrelation between m/z of glycopeptides and the optimum CID energyvalues means that an estimated value of optimum CID energy for any typeof test material can be calculated on the basis of this approximatecurve.

Next, the oxonium ion of m/z=138 in glycopeptides was measured at theoptimum CID energy by multiple reaction monitoring mode while using aglycopeptide whose mass is known as a standard, and detectionsensitivity of the glycopeptides was examined. As a result, thedetection sensitivity of 30 attomol (injection amount) was observed intwo types of isolated glycopeptides and there observed linearity ofsignal intensities in a wide range from 30 attomol to 30 femtomol (FIG.5). This result shows that the method of the present invention in whichthe oxonium ion of m/z=138 is measured at the optimum CID energyachieves measurement sensitivity and quantitative performance exceedingthose of a conventional method for glycopeptide measurement. Note thatthe value of R² in FIG. 5 indicates a multiple correlation coefficient.Note also that the same explanation as that for the schematic glycanstructures shown in FIG. 2 can be applied to the schematic glycanstructures shown in FIG. 5.

Example 4 Dual Monitoring of Glycoform in Fc and Fab Regions of Antibody

Site-specific glycan structure analysis of a glycoprotein was attemptedwhich analysis combines oxonium ion (m/z=138) monitoring andenergy-resolved oxonium ion profiling for quantification andverification of each structure, respectively. As a test material to beanalyzed, cetuximab (Merck Serono Co., Ltd.) which is a therapeuticantibody was used. The test material was denatured and digested in asimilar manner to Example 2. With the use of the OASIS HLB solid-phaseextraction cartridge, an undigested protein was removed from the testmaterial thus digested and then the test material was desalted andpurified. The peptide eluate was diluted and subjected to massspectrometry using a triple quadrupole mass spectrometer with nano-HPLC.In the triple quadrupole mass spectrometer, the oxonium ion (m/z=138)was measured for all currently predicted 40 types of glycoforms by theMRM mode (FIG. 6). In the chromatogram of FIG. 6, the horizontal axisrepresents a retention time in LC, and the vertical axis represents theintensity of the oxonium ion (m/z=138) which is expressed by the countnumber of the oxonium ion.

As shown in FIG. 6, a peak was observed within a range of retention timeof 13 to 14 minutes and within a range of retention time of 17 to 19minutes. The peak within the range of retention time of 13 to 14 minutescorresponds to a glycopeptide (amino acid sequence: EEQYN₂₉₇STYR, SEQ IDNO: 1) in the Fc region, and the peak within the range of retention timeof 17 to 19 minutes corresponds to a Fab glycopeptide (amino acidsequence: MNSLQSN₈₈DTAIYYCAR, SEQ ID NO: 2). It is thus possible tosimultaneously analyze, by a single analysis, two types of glycopeptidesthat are different from each other in glycosylation site.

Although the retention time in reversed-phase HPLC is mainly determinedby an amino acid composition, there observed, in the present analysismethod, a wide range of retention time arising solely from glycoformdifference, including baseline separation of glycopeptides which havealmost identical molecular weight. For example, Neu5Gc has molecularweight 307 Da and is smaller only by 1 Da than combined mass of hexoseand fucose (308 Da). Therefore glycopeptides whose predicted structurecontain Neu5Gc often result in two peaks on MRM chromatogram (FIG. 7)due to crosstalk with similar glycopeptide with Neu5Gc substituted withhexose and fucose.

The present analysis led to discovery of hyperfucosylated glycanstructures. Further, it was found out that hyperfucosylated form wasabsent for hypergalactosylated or disialylated structures, and it wastherefore speculated that extra fucosylation occurs on antennary GlcNAc(a branch at a non-reducing terminal of a core structure), which forms aLewis×motif, in a competitive manner with terminal galactosylation(α-Gal) or sialylation (Neu5Gc) for unmodified antenna.

To demonstrate this, structures of antennas in a biantennary structure,which has two antennas, and a triantennary structure, which has threeantennas, were analyzed by quantification using oxonium ion (m/z=138)monitoring and by structure verification using an energy-resolvedoxonium ion profile. The result is shown in Table 2.

TABLE 2 Composition α- % Abundance Gal Lewis X Neu5Gc BiantennaryTriantennary Antenna 1 — — 6.4 1.1 occupancy = 1 — 1 — 0.7 0.2 — — 1 7.10.9 Antenna 2 — — 25.9 1.7 occupancy = 2 1 1 — 2.5 1.0 1 — 1 18.5 1.3 —2 — 0.2 0.1 — 1 1 1.0 0.2 — — 2 3.3 0.3 Antenna 3 — — — 2.5 occupancy =3 2 1 — — 1.0 2 — 1 — 1.8 1 2 — — 0.2 1 1 1 — 0.5 1 — 2 — 0.6 — 3 — —0.0 — 2 1 — 0.1 — 1 2 — 0.2 — — 3 — N.D. N.D. = Not Detected

In Table 2, the “α-Gal” column shows the number of galactosylatedantennas, the “Lewis X” column shows the number of antennas withLewis×motif, and the “Nue5Gc” column shows the number of sialylatedantennas. The “biantennary” column shows percentages of those ofexamined biantennary structures which had the glycan structure(s) shownon the left side in a corresponding row, and the “triantennary” columnshows percentages of those of examined triantennary structures which hadthe glycan structure(s) shown on the left side in a corresponding row.For example, out of the examined biantennary structures, percentage ofbiantennary structures whose one antenna was galactosylated and otherantenna was sialylated was 18.5%.

As a result, in the biantennary structures in which only one antenna wasmodified, galactosylation, sialylation, and formation of a Lewis×motifoccurred at a ratio of 46:50:5. This is estimated to reflect therelative catalytic rates of glycosyltransferases. However, in thebianntennary structures in which both of the antennas were modified,relative abundances of a second antenna exhibited a different patternfrom a first antenna, tending strongly to galactosylation. This suggeststhat modification of the first antenna affected the reactivity of thesecond antenna.

FIG. 8 shows energy-resolved oxonium ion profiles of two typesglycopeptides containing Lewis×motif. In the energy-resolved oxonium ionprofiles, an oxonium ion of m/z=290 and an oxonium ion of m/z=512 werealso detected and plotted. The same explanation as that for theschematic glycan structures shown in FIG. 2 can be applied to theschematic glycan structures shown in FIG. 8. FIG. 8 shows thatstructures predicted to contain Lewis×motif were signified by high-leveldetection of the oxonium ion of m/z=512 which corresponds to[Hex+GlcNAc+Fuc] oxonium ion.

Other features of cetuximab revealed in this analysis were in accordancewith previous reports. For example, the most major glycoform in Fcglycan was an non-galactosylated biantennary structure, and relativelyhigh level of hybrid and high-mannose structures were also detected.

It was thus demonstrated that the present analysis method was capable ofmonitoring glycan heterogeneity of both Fc and Fab regions with highaccuracy and high sensitivity.

Example 5 Lot-to-Lot Heterogeneity of Glycoform of Therapeutic Antibody

Example 5 evaluated the analysis method of the above embodiment forapplicability to lot-to-lot quality control analysis of glycanheterogeneity, by using four non-continuous lots of trastuzumab (ChugaiPharmaceutical Co., Ltd.) and four non-continuous lots of bevacizumab(Chugai Pharmaceutical Co., Ltd.).

The test materials were denatured, digested, and purified in a similarmanner to cetuximab in Example 2, and were then subjected to massspectrometry using a triple quadrupole mass spectrometer with anano-HPLC, so as to verify and quantify glycan structures. In the massspectrometry, glycopeptides having the amino acid sequence representedby SEQ ID NO: 1 were measured. The lots were numbered in a productionorder. Analysis was performed three times for each lot, and statisticalsignificance of a difference between two consecutive lots was evaluatedby Student's t-test. The result is shown in FIG. 9. (a) of FIG. 9 showsa result concerning trastuzumab, and (b) of FIG. 9 shows a resultconcerning bevacizumab. In both of (a) and (b) of FIG. 9, abundanceratios of three types of glycan structures are shown. The sameexplanation as that for the schematic glycan structures shown in FIG. 2can be applied to the schematic glycan structures shown in FIG. 9.

As shown in FIG. 9, there observed a significant difference in abundanceratios of the glycan structures between Lot1 and Lot2. Meanwhile, inboth of the antibodies, there observed almost no difference between Lot2and Lot3. However, there observed again a significant difference betweenLot3 and Lot4. In FIG. 9, the “*” marks each indicate p<0.05 in theStudent's t-test. A glycoform that was most prone to lot-to-lotvariability was the frequency of galactosylation onto GlcNAc in abiantennary structure. This means that either an enzyme expression levelor a rate of antibody production can change by environmental factors. Adecrease in abundance of galactosylated form was clearly compensated byan increase in abundance of non-galactosylated form. Moreover, as aresult of observation, bevacizumab contained a non-glycosylated peptide(peptide consisting of the amino acid sequence represented by SEQ IDNO: 1) and that an amount of this peptide is also prone to variation.Because glycopeptide detection by oxonium ion (m/z=138) monitoring wasabout 10-fold more sensitive than detection of a non-glycosylatedcounterpart, actual percentage of non-glycosylated counterpart peptidecould be as high as a few percent and deserves good attention forquality control.

Example 6 Analysis of Glycopeptide Having O-Glycan

In Example 6, a Human Transferrin-derived glycopeptide having anO-glycan was analyzed.

Similar processes to those for human immunoglobulin (IgG) in Example 1were carried out up to the tryptic digestion. After the trypticdigestion, a mixture thus prepared was heated at 100° C. for 10 minutesso as to inactivate trypsin. Thereto, proline specific endopeptidase(Toyobo Co., Ltd.) whose amount is 1/20 (w/w) of Human Transferrin usedfor the digestion was added. After reaction at 37° C. for 8 hours,acetonitrile which is three times in volume (v/v) was added so as toobtain 75% acetonitril solution. This solution was desalted byhydrophillic solid-phase extraction plate HILIC μElution (Nihon WatersK.K.). In this desalting process, 100 mM of a triethylammoniumbicarbonate 75% acetonitrile solution was used as a solid phaseequilibrium and washing buffer, and 100 mM of a triethylammoniumbicarbonate 25% acetonitrile solution was used as an extraction bufferfrom a solid phase. An eluate thus obtained was dried to solid bySpeedvac, and was then redissolved in ultrapure water. A solution thusobtained was used as a measurement test material to be subjected to massspectrometry. Measurement and data processing of oxonium ions werecarried out for a plurality of transitions in which m/z of precursorions was set to 991.4 and only the CID energy was changed as inExample 1. The glycopeptide which is the measurement test material issummarized as follows: amino acid sequence: S₅₂DGPSVACVK (SEQ ID NO: 3,Ser52 is a glycosylation site), glycan composition: HexNAc×3, Hex×2,Fuc×1, Neu5AC×3 (composition which an N-glycan nerver has), peptide mass(MW): 1018.5, glycan mass (MW): 1970.7, glycopeptide mass (MW): 2971.2(−18Da because of β-glycan linkage), measurement ion: m/z=991.4(3+).

As a result, the energy-resolved oxonium ion profile shown in FIG. 10was obtained. The energy-resolved oxonium ion profile shown in FIG. 10is utterly different from that obtained from an N-glycopeptide havingsialic acid. It is thus possible to obtain higher-level information ofeven a glycan structure of an O-glycopeptide that is greatly differentfrom an N-glycopeptide in form of a glycan structure. Further, it wasconfirmed that, also in an O-glycopeptide, generation efficiency of m/zbecomes maximum at optimum CID energy estimated from m/z of theglycopeptide of Example 6 with the use of the linear correlation(Example 2) between m/z of glycopeptides and the optimum CID energyvalue.

INDUSTRIAL APPLICABILITY

The present invention is applicable to analysis of a glycan structure ofa glycoprotein, and is, for example, suitably applicable in qualitycontrol tests of glycan structures of biotechnology-based drugs such astherapeutic antibodies.

1. A method for analyzing a glycan structure of a test material having aglycan, comprising the steps of: (a) measuring specific types of productions produced from the test material at various values of CID energy byMS/MS; (b) creating an energy-resolved profile including yield curvesrepresenting relationships between (i) the values of the CID energy and(ii) measured amounts of the respective specific types of product ions;(c) preparing a reference profile including yield curves representingrelationships between (i) the values of the CID energy and (ii) measuredamounts of respective same types of product ions produced from areference test material as the specific types of product ions, thereference test material being a test material having a glycan and whosestructure is known; and (d) identifying the glycan structure of the testmaterial by comparing the energy-resolved profile obtained in the step(b) with the reference profile, the specific types of product ionsincluding at least two types of product ions derived from a protonatedmonosaccharide or disaccharide, and in the step (a), the measurement byMS/MS being carried out by use of a mass spectrometer which causes noLow-mass cutoff.
 2. The method according to claim 1, wherein thespecific types of product ions include at least two types of productions selected from the group consisting of product ions having m/z of163, 168, 186, 204, 274, 290, 292, 308, 366, 454, and 470, respectively.3. The method according to claim 2, wherein the specific types ofproduct ions include product ions having m/z of 163, 204, 274, and 366,respectively.
 4. The method according to claim 1, wherein: the specifictypes of product ions further include a product ion having m/z of 138;and in the step (b), the energy-resolved profile is created bynormalizing the yield curves with use of a measured amount of theproduct ion having m/z of
 138. 5. The method according to claim 1,wherein: in the step (a), the measuring is performed by using a samplein which a standard material which has a glycan and whose concentrationis known is added in addition to the test material; the specific typesof product ions include a product ion having m/z of 138; and the methodfurther comprises the step of (e) quantifying the test material bycomparing (i) a measured amount of the product ion having m/z of 138produced from the test material and (ii) a measured amount of theproduct ion having m/z of 138 produced from the standard material. 6.The method according to claim 5, wherein in the step (e), the testmaterial is quantified on basis of (i) the measured amount of theproduct ion having m/z of 138 produced from the test material and (ii)the measured amount of the product ion having m/z of 138 produced fromthe standard material, each of which measured amounts are obtained at avalue of the CID energy at which the product ion having m/z of 138becomes maximum.
 7. The method according to claim 6, wherein the valueof the CID energy at which the product ion having m/z of 138 becomesmaximum is a value estimated based on a calibration curve and a value ofm/z of a precursor ion of the test material to be analyzed, thecalibration curve being created by (I) measuring, at various values ofthe CID energy, in advance the product ion having m/z of 138 in aplurality of test materials each having a glycan by MS/MS and then (II)carrying out linear regression analysis with use of (i) values of theCID energy at which measured amounts of the product ion having m/z of138 in the plurality of test materials become maximum and (ii) values ofm/z of precursor ions of the plurality of test materials.
 8. The methodaccording to claim 1, wherein in the step (a), the measurement by MS/MSis carried out by use of a triple quadrupole mass spectrometer.
 9. Themethod according to claim 1, wherein the test material is aglycopeptide.
 10. The method according to claim 9, wherein theglycopeptide is a glycopeptide having an N-glycan.
 11. The methodaccording to claim 9, wherein the glycopeptide is a glycopeptide havingan O-glycan.